Coupling of the nucleotide P2Y 4 receptor to neuronal ion channels

Abstract

1 G protein-linked P2Y nucleotide receptors are known commonly to stimulate the phosphoinositide signalling pathway. However, we have previously demonstrated that the cloned P2Y 2, P2Y 6 and P2Y 1 receptors couple to neuronal N-type Ca 2+ channels and to M-type K + channels. Here we investigate the coupling of recombinant, neuronally expressed rat- and human P2Y 4 receptors (rP2Y 4, hP2Y 4) to those channels. 2 Rat sympathetic neurones were nuclear-injected with a P2Y 4 cDNA plasmid. A subsequent activation of rP2Y 4 or hP2Y 4 by UTP (100 μM) in whole-cell (ruptured-patch) mode produced only about 12% inhibition of the N-type Ca 2+ current (I cA(N)). Surprisingly, in perforated patch mode, UTP produced much more inhibition of I Ca(N) (maximally 51%), with an IC 50 value of 273 nM. This inhibition was voltage-dependent and was blocked by co-expression of the βγ-binding transducin Gα-subunit. Pertussis toxin (PTX) pretreatment also suppressed I Ca(N) inhibition. 3 UTP inhibited the M-current, recorded in perforated patch mode, by (maximally) 52%, with I C50 values of 21 nM for rP2Y 4 and 28 nM for hP2Y 4. This inhibition was not affected by PTX pretreatment. 4 With rP2Y 4, ATP inhibited the M-current (IC 50 524 nM, 26 times weaker than UTP), whereas ATP had no agonist activity at hP2Y 4. This suggests a difference in agonist binding site between rP2Y 4 and hP2Y 4. 5 We conclude that, in contrast to other nucleotide receptors studied, the P2Y 4 receptor couples much more effectively to M-type K + channels than to Ca 2+ channels. Coupling to the Ca 2+ channels involves the βγ-subunits of G i/o-proteins and requires a diffusible intracellular component that is lost in ruptured-patch recording.